Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the ...precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A number of stresses, including nutrient stress, temperature shock, DNA damage, and hypoxia, can lead to changes in gene expression patterns caused by a general shutdown and reprogramming of protein ...synthesis. Each of these stress conditions results in selective recruitment of ribosomes to mRNAs whose protein products are required for responding to stress. This recruitment is regulated by elements within the 5′ and 3′ untranslated regions of mRNAs, including internal ribosome entry segments, upstream open reading frames, and microRNA target sites. These elements can act singly or in combination and are themselves regulated by trans-acting factors. Translational reprogramming can result in increased life span, and conversely, deregulation of these translation pathways is associated with disease including cancer and diabetes.
Post-transcriptional control makes a major contribution to the overall regulation of gene expression pathway. Within the cytoplasm this is mediated by a combination of regulatory RNA motifs within ...the 5′ and 3′ untranslated regions of mRNAs and their interacting protein/RNA partners. One of the most common regulatory RNA elements in mammalian transcripts (present in approximately 40% of all mRNAs) are upstream open reading frames (uORFs). However, despite the prevalence of these RNA elements how they function is not well understood. In general, they act to repress translation of the physiological ORF under control conditions, and under certain pathophysiological stresses this repression can be alleviated. It is known that re-initiation following the translation of an uORF is utilised in some situations however there are numerous alternative mechanisms that control the synthesis of a protein whose mRNA contains uORFs. Moreover, the trans-acting factors that are also involved in this process are not well defined. In this review we summarise our current understanding of this area and highlight some common features of these RNA motifs that have been discovered to date.
Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long ...noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.
During prion disease, an increase in misfolded prion protein (PrP) generated by prion replication leads to sustained overactivation of the branch of the unfolded protein response (UPR) that controls ...the initiation of protein synthesis. This results in persistent repression of translation, resulting in the loss of critical proteins that leads to synaptic failure and neuronal death. We have previously reported that localized genetic manipulation of this pathway rescues shutdown of translation and prevents neurodegeneration in a mouse model of prion disease, suggesting that pharmacological inhibition of this pathway might be of therapeutic benefit. We show that oral treatment with a specific inhibitor of the kinase PERK (protein kinase RNA-like endoplasmic reticulum kinase), a key mediator of this UPR pathway, prevented UPR-mediated translational repression and abrogated development of clinical prion disease in mice, with neuroprotection observed throughout the mouse brain. This was the case for animals treated both at the preclinical stage and also later in disease when behavioral signs had emerged. Critically, the compound acts downstream and independently of the primary pathogenic process of prion replication and is effective despite continuing accumulation of misfolded PrP. These data suggest that PERK, and other members of this pathway, may be new therapeutic targets for developing drugs against prion disease or other neurodegenerative diseases where the UPR has been implicated.
Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. ...Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A growing body of work demonstrates the importance of post-transcriptional control, in particular translation initiation, in the overall regulation of gene expression. Here we focus on the ...contribution of regulatory elements within the 5' and 3' untranslated regions of mRNA to gene expression in eukaryotic cells including terminal oligopyrimidine tracts, internal ribosome entry segments, upstream open reading frames and cytoplasmic polyadenylation elements. These mRNA regulatory elements may adopt complex secondary structures and/or contain sequence motifs that allow their interaction with a variety of regulatory proteins, RNAs and RNA binding proteins, particularly hnRNPs. The resulting interactions are context-sensitive, and provide cells with a sensitive and fast response to cellular signals such as hormone exposure or cytotoxic stress. Importantly, an increasing number of diseases have been identified, particularly cancers and those associated with neurodegeneration, which originate either from mutation of these regulatory motifs, or from deregulation of their cognate binding partners.
PTB (polypyrimidine-tract-binding protein) is a ubiquitous RNA-binding protein. It was originally identified as a protein with a role in splicing but it is now known to function in a large number of ...diverse cellular processes including polyadenylation, mRNA stability and translation initiation. Specificity of PTB function is achieved by a combination of changes in the cellular localization of this protein (its ability to shuttle from the nucleus to the cytoplasm is tightly controlled) and its interaction with additional proteins. These differences in location and trans-acting factor requirements account for the fact that PTB acts both as a suppressor of splicing and an activator of translation. In the latter case, the role of PTB in translation has been studied extensively and it appears that this protein is required for an alternative form of translation initiation that is mediated by a large RNA structural element termed an IRES (internal ribosome entry site) that allows the synthesis of picornaviral proteins and cellular proteins that function to control cell growth and cell death. In the present review, we discuss how PTB regulates these disparate processes.
The ability of mammalian cells to modulate global protein synthesis in response to cellular stress is essential for cell survival. While control of protein synthesis is mediated by the regulation of ...eukaryotic initiation and elongation factors, RNA-binding proteins (RBPs) provide a crucial additional layer to post-transcriptional regulation. RBPs bind specific RNA through conserved RNA-binding domains and ensure that the information contained within the genome and transcribed in the form of RNA is exported to the cytoplasm, chemically modified, and translated prior to folding into a functional protein. Thus, this group of proteins, through mediating translational reprogramming, spatial reorganisation, and chemical modification of RNA molecules, have a major influence on the robust cellular response to external stress and toxic injury.
Protein synthesis is a tightly regulated process that enables post-transcriptional control of gene expression. Dysregulation of this process is associated with the development and progression of ...cancers because components of the translational machinery function at the point of convergence of aberrant cell signaling pathways. Drugs designed to inhibit mRNA translation are currently in preclinical and early clinical development, and are likely to provide effective anticancer strategies in the future. In this Review, we summarize the main components of translation and describe how alterations in these proteins and their principle upstream signaling pathways can impact on cancer. The first inhibitors of translation, drugs designed to target eIF4E, have been trialed in hematologic malignancies, while antisense oligonucleotides against eIF4E are also due to enter clinical trials. Here, we discuss the mode of action of drugs designed to inhibit mRNA translation and other promising therapies that are in preclinical development with the aim of becoming anticancer agents.
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